The application is for support to continue studies of the structure, mechanism, and regulation of proton-translocating ATPases in the V-type family. The V-type ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macromolecular processing, and degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also play a role in renal acidification, bone resorption, and tumor metastasis. Understanding how V-ATPases are regulated is important to understanding these processes. This laboratory has previously shown that the V-ATPase from clathrin- coated vesicles is organized into a peripheral V1 domain responsible for ATP hydrolysis and an integral Vo domain responsible for proton translocation. Chemical modification has been used to probe the structure of the nucleotide binding sites, this group has suggested that disulfide bond formation may play a role in regulation of V-ATPase activity in vivo. Reassembly studies have been used to test the function of individual subunits, including a protein shared between the V-ATPase and AP-2 adaptor complexes. More recently, they have begun mutagenesis studies of the yeast V-ATPase to identify residues important in V-ATPase activity. Four specific aims will be pursued in the proposed studies. To determine the structure and function of the noncatalytic nucleotide binding sites on the B subunit, cysteine-scanning mutagenesis will be employed. The role of B subunit isoforms in activity and intracellular targeting of V-ATPases will also be tested. To further define the structure of the catalytic A subunit, residues participating in nucleotide binding will be identified by site-directed mutagenesis. The proximity and role of conserved A subunit cysteine residues in regulation of vacuolar acidification will also be further probed. Studies of the 100 kDa subunit will focus on identification of second-site suppressors of mutations demonstrated to affect function, on elucidation of the topography of the 100 kDa subunit, and on identification of mutations which confer concanamycin resistance. Finally, the arrangement and function of accessory subunits, including AP50 and the VMA6 gene product, will be investigated. These studies should provide further insight into the structure and regulation of this important family of H+-ATPase.